Antibodies against GPR64 and uses thereof
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example 1
GPR64 Gene Expression Profiling in Ovarian Cancer and Normal Tissues
[0152]This example describes the use of GeneChip expression profiling to identify GPR64 as a valid ovarian cancer target.
SUMMARY
[0153]Gene expression of 66 ovarian cancer samples was compared to 347 normal adult tissues representing 58 different organs. The goal was to look for genes that are up-regulated in ovarian cancer and are localized to the cell surface for antibody accessibility, but have little to no expression in vital organs to minimize undesirable side effects of a therapeutic antibody. Genes with the desired expression profile were examined by extensive bioinformatic analysis to determine their structural and functional classification, and determine their potential for cell surface localization. The GPR64 gene (National Center for Biotechnology Information reference sequence no. NM—005756.1;Ref. Osterhoffet al., 1997, DNA Cell Biol. 16:379-389) displayed all the desired characteristics.
[0154]RNA Extract...
example 2
Anti-GPR64 Inhibits Tumor Cell Growth In Vivo
[0159]The following example illustrates that GPR64 antibodies are effective at reducing tumor volume in vivo.
[0160]Animal studies were conducted using SCID mice immunized with the Human tumor cell line NCI-H460. The NCI-H460 cell line expresses the antigen recognized by antibody GPR64-18. The VH and VL nucleotide and amino acid sequences of antibody GPR64-18 are provided in FIG. 2 (SEQ ID NOs:7,8,17,18).
[0161]The antibodies were made via standard methods using a fusion protein between the large N-terminus of GPR64 and human Fc as the immunogen.
[0162]To initiate tumor growth in vivo SCID mice were injected with the NCI-H460 tumor cell line and tumors were allowed to grow. When tumors reach a size of between 50-100 mm3, animals were distributed into groups and subjected to treatment with either:[0163]a.) an isotype control antibody;[0164]b.) one of the five GPR64 antibodies, whose VH and VL sequences depicted in FIG. 2 (SEQ ID NOs:2-22), or...
example 3
GPR64 Knockdown by RNAi Inhibits Cell Proliferation of GPR64 Expressing Cancer Cells
[0171]The following example illustrates that GPR64 expression is essential for tumor cell growth in vitro and validates GPR64 as an ovarian cancer target.
[0172]Proteins can be down-regulated using short interfering double stranded RNAs (siRNA) specific to the cognate mRNA of the protein of interest. This approach was used to show that inhibition of GPR64 down-regulates cell growth and causes cell death. Thus, these experiments are consistent with the results of the experiments described in Example 2 and thereby confirm the basic conclusion that down-regulation of GPR64 expression will provide an effective treatment for cancers involving GPR64 protein expression.
RNAi Assay Method
[0173]Plasmids encoding an siRNA specific for GPR64, or a control siRNA, were introduced into two GPR64+human tumor cell lines that require GPR64 for growth: H460, Me180. In addition, the same siRNAs were introduced into GPR64...
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